Poly(N-methyl-N-vinylacetamide): A Strong Alternative to PEG for Lipid-Based Nanocarriers Delivering siRNA.

Lipid-based nanocarriers have demonstrated high interest in delivering genetic material, exemplified by the success of Onpattro and COVID-19 vaccines. While PEGylation imparts stealth properties, it hampers cellular uptake and endosomal escape, and may trigger adverse reactions like accelerated blood clearance (ABC) and hypersensitivity reactions (HSR). This work highlights the great potential of amphiphilic poly(N-methyl-N-vinylacetamide) (PNMVA) derivatives as alternatives to lipid-PEG for siRNA delivery. PNMVA compounds with different degrees of polymerization and hydrophobic segments, are synthesized. Among them, DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)-PNMVA efficiently integrates into lipoplexes and LNP membranes and prevents protein corona formation around these lipid carriers, exhibiting stealth properties comparable to DSPE-PEG. However, unlike DSPE-PEG, DSPE-PNMVA24 shows no adverse impact on lipoplexes cell uptake and endosomal escape. In in vivo study with mice, DSPE-PNMVA24 lipoplexes demonstrate no liver accumulation, indicating good stealth properties, extended circulation time after a second dose, reduced immunological reaction, and no systemic pro-inflammatory response. Safety of DSPE-PNMVA24 is confirmed at the cellular level and in animal models of zebrafish and mice. Overall, DSPE-PNMVA is an advantageous substitute to DSPE-PEG for siRNA delivery, offering comparable stealth and toxicity properties while improving efficacy of the lipid-based carriers by minimizing the dilemma effect and reducing immunological reactions, meaning no ABC or HSR effects.

Poly(vinyl pyrrolidone) derivatives as PEG alternatives for stealth, non-toxic and less immunogenic siRNA-containing lipoplex delivery.

The recent approval of Onpattro® and COVID-19 vaccines has highlighted the value of lipid nanoparticles (LNPs) for the delivery of genetic material. If it is known that PEGylation is crucial to confer stealth properties to LNPs, it is also known that PEGylation is responsible for the decrease of the cellular uptake and endosomal escape and for the production of anti-PEG antibodies inducing accelerated blood clearance (ABC) and hypersensitivity reactions. Today, the development of PEG alternatives is crucial. Poly(N-vinyl pyrrolidone) (PNVP) has shown promising results for liposome decoration but has never been tested for the delivery of nucleic acids. Our aim is to develop a series of amphiphilic PNVP compounds to replace lipids-PEG for the post-insertion of lipoplexes dedicated to siRNA delivery. PNVP compounds with different degrees of polymerization and hydrophobic segments, such as octadecyl, dioctadecyl and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), were generated. Based on the physicochemical properties and the efficiency to reduce protein corona formation, we showed that the DSPE segment is essential for the integration into the lipoplexes. Lipoplexes post-grafted with 15% DSPE-PNVP30 resulted in gene silencing efficiency close to that of lipoplexes grafted with 15% DSPE-PEG. Finally, an in vivo study in mice confirmed the stealth properties of DSPE-PNVP30 lipoplexes as well as a lower immune response ABC effect compared to DSPE-PEG lipoplexes. Furthermore, we showed a lower immune response after the second injection with DSPE-PNVP30 lipoplexes compared to DSPE-PEG lipoplexes. All these observations suggest that DSPE-PNVP30 appears to be a promising alternative to PEG, with no toxicity, good stealth properties and lower immunological response.

Dimeric polyphenols to pave the way for new antimalarial drugs.

Because of the threat of resistant Plasmodium sp., new orally active antimalarials are urgently needed. Inspired by the structure of ellagic acid, exhibiting potent in vivo and in vitro antiplasmodial effects, polyphenolic structures possessing a similar activity-safety profile were synthesized. Indeed, most exhibited a marked in vitro effect (IC50 < 4 µM) on resistant P. falciparum, without any detrimental effects reported during the toxicity assays (hemolysis, cytotoxicity, in vivo). In addition, they possessed a greater hydrosolubility (from 7 µM to 2.7 mM) compared to ellagic acid. Among them, 30 is the most promising for antimalarial purposes since it displayed a significant parasitaemia reduction after oral administration in mice (50 mg kg-1) compared to the orally ineffective ellagic acid. In conclusion, our investigations led to the identification of a promising scaffold, which could bring new insights for malaria treatment.

Broadening the phenotypic spectrum and physiological insights related to EIF2S3 variants.

Mental deficiency, epilepsy, hypogonadism, microcephaly, and obesity syndrome is a severe X-linked syndrome caused by pathogenic variants in EIF2S3. The gene encodes the γ subunit of the eukaryotic translation initiation factor-2, eIF2, essential for protein translation. A recurrent frameshift variant is described in severely affected patients while missense variants usually cause a moderate phenotype. We identified a novel missense variant (c.433A>G, p.(Met145Val)) in EIF2S3 in a mildly affected patient. Studies on zebrafish confirm the pathogenicity of this novel variant and three previously published missense variants. CRISPR/Cas9 knockout of eif2s3 in zebrafish embryos recapitulate the human microcephaly and show increased neuronal cell death. Abnormal high glucose levels were identified in mutant embryos, caused by beta cell and pancreatic progenitor deficiency, not related to apoptosis. Additional studies in patient-derived fibroblasts did not reveal apoptosis. Our results provide new insights into disease physiopathology, suggesting tissue-dependent mechanisms.

Antimalarial Activities of Alkyl Cyclohexenone Derivatives Isolated from the Leaves of Poupartia borbonica.

Bioactivity-guided fractionation of the ethyl acetate extract of the leaves of Poupartia borbonica led to the isolation of three new alkyl cyclohexenone derivatives 1-3, and named Poupartone A-C. The structures of the new compounds were elucidated by 1D and 2D NMR spectroscopic data analysis and MS, whereas calculated and experimental ECD spectra were used to define the absolute configurations. These compounds were active against 3D7 and W2 Plasmodium falciparum strains with IC50 values between 0.55 and 1.81 µM. In vitro cytotoxicity against WI38 human fibroblasts and the human cervical cancer cell line HeLa (WST-1 assay) showed that these compounds were also cytotoxic, but no hemolytic activity was observed for the extract and pure compounds. An in vivo antimalarial assay was performed on the major cyclohexenone using P. berghei-infected mice at a dose of 15 mg/kg/day ip. The assay revealed growth inhibition of 59.1 and 69.5% at days 5 and 7 postinfection, respectively, although some toxicity was observed. Zebrafish larvae were used as a model to determine the type of toxicity, and the results showed cardiac toxicity. The methanol extract was also studied, and it displayed moderate antiplasmodial properties in vitro. This extract contained the known flavonoids, quercetin, 3'-O-hydroxysulfonylquercetin, quercitrin, and isoquercitrin as well as ellagic acid, which showed high to low activity against the 3D7 P. falciparum strain.

Two novel EIF2S3 mutations associated with syndromic intellectual disability with severe microcephaly, growth retardation, and epilepsy.

X-chromosome exome sequencing was performed to identify the genetic cause of syndromic intellectual disability in two unrelated families with suspected X-linked inheritance. In both families, affected males presented with severe intellectual disability, microcephaly, growth retardation, and epilepsy. A missense mutation (c.777T>G p.(Ile259Met)) and a frameshift mutation (c.1394_1397del p.(Ile465Serfs*4)) were identified in the EIF2S3 gene in the hemizygous state in affected patients, and in the heterozygous states female obligate carriers. A missense mutation in EIF2S3, coding for the gamma-subunit of the translation initiation factor eIF2, was reported once in a family presenting with similar clinical features. Morpholino-based knockdown of the zebrafish EIF2S3 ortholog (eif2s3) recapitulates the human microcephaly and short stature phenotype, supporting the pathogenicity of the identified variants. Our data confirm that EIF2S3 mutation is implicated in a rare, but recognizable, form of syndromic intellectual disability. © 2016 Wiley Periodicals, Inc.

Automating ChIP-seq experiments to generate epigenetic profiles on 10,000 HeLa cells.

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples.

Dre-miR-2188 targets Nrp2a and mediates proper intersegmental vessel development in zebrafish embryos.

BACKGROUND: MicroRNAs (miRNAs) are a class of small RNAs that are implicated in the control of eukaryotic gene expression by binding to the 3'UTR of target mRNAs. Several algorithms have been developed for miRNA target prediction however, experimental validation is still essential for the correct identification of miRNA targets. We have recently predicted that Neuropilin2a (Nrp2a), a vascular endothelial growth factor receptor which is essential for normal developmental angiogenesis in zebrafish, is a dre-miR-2188 target. METHODOLOGY: Here we show that dre-miR-2188 targets the 3'-untranslated region (3'UTR) of Nrp2a mRNA and is implicated in proper intersegmental vessel development in vivo. Over expression of miR-2188 in zebrafish embryos down regulates Nrp2a expression and results in intersegmental vessel disruption, while its silencing increases Nrp2a expression and intersegmental vessel sprouting. An in vivo GFP sensor assay based on a fusion between the GFP coding region and the Nrp2a 3'UTR confirms that miR-2188 binds to the 3'UTR of Nrp2a and inhibits protein translation. CONCLUSIONS: We demonstrate that miR-2188 targets Nrp2a and affects intersegmental vessel development in zebrafish embryos.

Matrix metalloproteinase-2 governs lymphatic vessel formation as an interstitial collagenase.

Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)-2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2 knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis.

Zebrafish Sox7 and Sox18 function together to control arterial-venous identity.

Sox7 and Sox18 are members of the F-subgroup of Sox transcription factors family and are mostly expressed in endothelial compartments. In humans, dominant mutations in Sox18 are the underlying cause of the severe hypotrichosis-lymphedema-telangiectasia disorder characterized by vascular defects. However little is known about which vasculogenic processes Sox7 and Sox18 regulate in vivo. We cloned the orthologs of Sox7 and Sox18 in zebrafish, analysed their expression pattern and performed functional analyses. Both genes are expressed in the lateral plate mesoderm during somitogenesis. At later stages, Sox18 is expressed in all axial vessels whereas Sox7 expression is mainly restricted to the dorsal aorta. Knockdown of Sox7 or Sox18 alone failed to reveal any phenotype. In contrast, blocking the two genes simultaneously led to embryos displaying dysmorphogenesis of the proximal aorta and arteriovenous shunts, all of which can account for the lack of circulation observed in the trunk and tail. Gene expression analyses performed with general endothelial markers on double morphants revealed that Sox7 and Sox18 are dispensable for the initial specification and positioning of the major trunk vessels. However, morphants display ectopic expression of the venous Flt4 marker in the dorsal aorta and a concomitant reduction of the artery-specific markers EphrinB2a and Gridlock. The striking similarities between the phenotype of Sox7/Sox18 morphants and Gridlock mutants strongly suggest that Sox7 and Sox18 control arterial-venous identity by regulating Gridlock expression.

Sesn1 is a novel gene for left-right asymmetry and mediating nodal signaling.

Remarkable progress has been made in understanding the molecular mechanisms underlying left-right asymmetry in vertebrate animal models but little is known on left-right axis formation in humans. Previously, we identified SESN1 (also known as PA26) as a candidate gene for heterotaxia by positional cloning of the breakpoint regions of a de novo translocation in a heterotaxia patient. In this study, we show by means of a zebrafish sesn1-knockdown model that Sesn1 is required for normal embryonic left-right determination. In this model, developmental defects and expression data of genes implicated in vertebrate left-right asymmetry indicate a role for Sesn1 in mediating Nodal signaling. In the lateral plate mesoderm, Nodal signaling plays a central role in left-right axis formation in vertebrates and is mediated by FoxH1 transcriptional induction. In line with this, we show that Sesn1 physically interacts with FoxH1 or a FoxH1-containing complex. Mutation analysis in a panel of 234 patients with isolated heterotaxia did not reveal mutations, indicating that these are only exceptional causes of human heterotaxia. In this study, we identify SESN1 as an indispensable gene for vertebrate left-right asymmetry and a new player in mediating Nodal signaling.

Evi1 is specifically expressed in the distal tubule and duct of the Xenopus pronephros and plays a role in its formation.

The ecotropic viral integration site 1 (Evi1) and related MEL1 (MDS1/Evi1-like gene 1) genes are zinc finger oncogenic transcription factors involved in myeloid leukaemia. Here, we show that in Xenopus, Evi1 and MEL1 have partially overlapping restricted embryonic expression profiles. Within the pronephros, Evi1 and MEL1 are sequentially expressed within the distal tubule and duct compartments, Evi1 transcription being detected prior to any sign of pronephric morphogenesis. In the pronephros of zebrafish embryos, Evi1 expression is restricted to the posterior portion of the duct, the anterior portion having characteristics of proximal tubules. In the Xenopus pronephros, Evi1 expression is upregulated by retinoid signaling and repressed by overexpression of xWT1 and by Notch signaling. Overexpression of Evi1 from late neurula stage specifically inhibits the expression of proximal tubule and glomus pronephric markers. We show that the first zinc finger and CtBP interaction domains are required for this activity. Overexpression of a hormone-inducible Evi1-VP16 antimorphic fusion with activation at neurula stage disrupts distal tubule and duct formation and expands the expression of glomus markers. Although overexpression of this construct also causes in many embryos a reduction of proximal tubule markers, embryos with expanded and ectopic staining have been also observed. Together, these data indicate that Evi1 plays a role in the proximo-distal patterning of the pronephros and suggest that it may do so by functioning as a CtBP dependent repressor.

The Na+/PO4 cotransporter SLC20A1 gene labels distinct restricted subdomains of the developing pronephros in Xenopus and zebrafish embryos.

The embryonic pronephric kidneys of Xenopus and zebrafish serve as models to study vertebrate nephrogenesis. Recently, multiple subdomains within the Xenopus pronephros have been defined based on the expression of several transport proteins. In contrast, very few studies on the expression of renal transporters have been conducted in zebrafish. We have recently shown that the anterior and posterior segments of the zebrafish pronephric duct may correspond to the proximal tubule and distal tubule/duct compartments of the Xenopus and higher vertebrate pronephros, respectively. Here, we report the embryonic expression pattern of the Na(+)/PO(4) cotransporter SLC20A1 (PiT1/Glvr-1) gene encoding a type III sodium-dependent phosphate cotransporter in Xenopus and zebrafish. In Xenopus, SLC20A1 mRNA is expressed in the somitic mesoderm and lower level of expression is detected in the neural tube, eye, and neural crest cells. From stage 25, SLC20A1 is also detectable in the developing pronephros where expression is restricted to the late portion of the distal pronephric tubules. In zebrafish, SLC20A1 is transcribed from mid-somitogenesis in the anterior part of the pronephros where its expression corresponds to the rostral portion of the expression of other proximal tubule-specific markers. Outside the pronephros, lower level of SLC20A1 expression is also observed in the posterior cardinal and caudal veins. Based on the SLC20A1 expression domain and that of other transporters, four segments have been defined within the zebrafish pronephros. Together, our data reveal that the zebrafish and Xenopus pronephros have non-identical proximo-distal organizations.

Cloning and embryonic expression of zebrafish PLAG genes.

PLAG transcription factors play important roles in oncogenesis. To date three members of this subfamily of zinc finger proteins have been identified in humans and mice: PLAG1, PLAGL1 and PLAGL2. In this study, we identified zebrafish orthologs of PLAG1 and PLAGL2 and a novel member of this family, PLAGX. We examined the temporal expression of these three genes by quantitative real time RT-PCR and found that all three genes are maternally provided, expressed at low level during early somitogenesis and, during late somitogenesis and beyond, PLAG expression increases to reach a plateau level around 5 dpf. Whole mount in situ experiments revealed that PLAG1, PLAGL2 and PLAGX display a similar pattern of expression characterized by a low ubiquitous expression overcame by high expression in some restricted compartments such as the ventricular zone of the brain, the pectoral fin buds, the developing pharyngeal arches and the axial vasculature. We show that this pattern resembles the one observed for the proliferative marker PCNA, suggesting that the PLAG genes are expressed more strongly in zones of active proliferation. This hypothesis was proven for the ventricular zone shown to be a highly proliferative zone using the anti-phosphohistone H3 antibody that detects cells in mitosis.

Microarray screening for target genes of the proto-oncogene PLAG1.

PLAG1 is a proto-oncogene whose ectopic expression can trigger the development of pleomorphic adenomas of the salivary glands and of lipoblastomas. As PLAG1 is a transcription factor, able to activate transcription through the binding to the consensus sequence GRGGC(N)(6-8)GGG, its ectopic expression presumably results in the deregulation of target genes, leading to uncontrolled cell proliferation. The identification of PLAG1 target genes is therefore a crucial step in understanding the molecular mechanisms involved in PLAG1-induced tumorigenesis. To this end, we analysed the changes in gene expression caused by the conditional induction of PLAG1 expression in fetal kidney 293 cell lines. Using oligonucleotide microarray analyses of about 12 000 genes, we consistently identified 47 genes induced and 12 genes repressed by PLAG1. One of the largest classes identified as upregulated PLAG1 targets consists of growth factors such as the insulin-like growth factor II and the cytokine-like factor 1. The in silico search for PLAG1 consensus sequences in the promoter of the upregulated genes reveals that a large proportion of them harbor several copies of the PLAG1-binding motif, suggesting that they represent direct PLAG1 targets. Our approach was complemented by the comparison of the expression profiles of pleomorphic adenomas induced by PLAG1 versus normal salivary glands. Concordance between these two sets of experiments pinpointed 12 genes that were significantly and consistently upregulated in pleomorphic adenomas and in PLAG1-expressing cells, identifying them as putative PLAG1 targets in these tumors.

Mnt loss triggers Myc transcription targets, proliferation, apoptosis, and transformation.

Myc oncoproteins are overexpressed in most cancers and are sufficient to accelerate cell proliferation and provoke transformation. However, in normal cells Myc also triggers apoptosis. All of the effects of Myc require its function as a transcription factor that dimerizes with Max. This complex induces genes containing CACGTG E-boxes, such as Ornithine decarboxylase (Odc), which harbors two of these elements. Here we report that in quiescent cells the Odc E-boxes are occupied by Max and Mnt, a putative Myc antagonist, and that this complex is displaced by Myc-Max complexes in proliferating cells. Knockdown of Mnt expression by stable retroviral RNA interference triggers many targets typical of the "Myc" response and provokes accelerated proliferation and apoptosis. Strikingly, these effects of Mnt knockdown are even manifest in cells lacking c-myc. Moreover, Mnt knockdown is sufficient to transform primary fibroblasts in conjunction with Ras. Therefore, Mnt behaves as a tumor suppressor. These findings support a model where Mnt represses Myc target genes and Myc functions as an oncogene by relieving Mnt-mediated repression.